EPO, a glycoprotein having the molecular weight of 30,000 to 34,000, is a factor that stimulates production and differentiation of red blood cells. This protein acts by binding to receptors on erythrocyte precursor cells to result in increase of calcium ion concentration in a cell, increase of DNA biosynthesis, and stimulation for the formation of hemoglobin and the like. This EPO can be used for the treatment of anemia from renal failure, anemia of a premature baby, anemia from hypothyroidism, anemia from malnutrition, etc. The clinical use of recombinant human EPO is on the increase. However, such use may cause some inconvenience and high costs because it should be administered on the average three times a week due to its short half-life. Thus, if the in vivo activity of EPO is maintained for a long time, the administration frequency of EPO may be greatly decreased.
Efficacy of EPO is proportional to in vivo half-life thereof. It is known that in vivo half-life of EPO is correlated to the content of sialic acid that is located at the terminal of carbohydrate chains of EPO. Therefore, efficacy of EPO is highly dependent on the presence of carbohydrate chains. Since the forms of carbohydrates appear differently depending on the kind of cells where EPO is expressed, the same glycoproteins may have different carbohydrate structure if they are expressed in different cells. Although it has been recently demonstrated that some bacteria can attach the carbohydrate chains, typical bacteria, for example E. coli, are known not to do. Proteins expressed in E. coli do not contain the carbohydrate chains, and thus, E. coli-derived EPO, which does not contain the carbohydrate chains, exhibits positive in vitro activity but no in vivo activity. It is because deglycosylated EPO is rapidly eliminated from the human body and has extremely short half-life. In conclusion, the carbohydrate chains play a very important role in the activity of EPO.
Many studies have been made to enhance the activity of EPO. The main approach is substitutions of some amino acids of EPO by mutagenesis on the EPO gene. For example, PCT/US94/09257, filed by Amgen and titled “Erythropoietin analogs,” discloses a method to increase in vivo half-life of EPO by increasing the carbohydrate contents through mutagenesis. Also, an attempt to increase in vivo half-life of EPO was made by formation of EPO dimer. See, A. J. Sytkowski et al., J.B.C. vol. 274, No. 35, pp24773–24778. Besides, another known method is to enhance in vivo activity of EPO by fusing new amino acids, peptides, or protein fragments with EPO molecule in the genetic engineering manner and increasing the carbohydrate content, i.e., sialic acid content of EPO. However, all amino acids, peptides, or heterogeneous protein fragments may not be used for this purpose. In most cases, such modifications may result in decrease or loss of inherent activity of protein and may cause a problem of antigenicity when used in vivo.
Although it is not related to EPO, fusion proteins or chimeric proteins have been studied, for example, for follicle stimulating hormone that is a kind of sex hormone. See, Furuhashi et al., 1995, Mol. Endocrinol. However, the methods have not been applied to the industry since protein modifications using a genetic engineering method have many risks. That is, in most cases, the target protein may not be readily obtained without professional skills, and on the contrary, the inherent activity of protein may be decreased or lost by the addition or substitution of new amino acids.